Feline infectious peritonitis: Newer findings from around the world (2)

Over 100 published articles have appeared in the world’s literature concerning FIP since my extensive review of FIP in 2009 (1). The following is a summary of significant findings from a portion of these published works.

Origin of FIPV (the FECV to FIPV mutation)

The debate over the origins of the FIP virus (FIPV) continues to some degree, but there is no doubt that FIPV arises as a mutant of the ubiquitous feline enteric coronavirus (FECV). Although FIPVs are virtually identical genetically to FECVs within the same environment (2-4), FIP causing mutations are nonetheless unique to each cat (2,3,5,16). The nature of the mutations that cause an FECV to change to an FIPV has been the topic of several recent publications. The 3c accessory gene mutations were the first to be implicated in FECV-to-FIPV conversion (reviewed 1) and these findings have been corroborated by additional studies (2,3). However, a group from the University of Utrecht, after sequencing the complete genomes of a large number of FIPVs and FECVs, found a second mutation that occurred only in FIPVs (5). This mutation consisted of one or more single nucleotide polymorphisms (SNPs) in the fusion domain of the spike (S) gene that caused minor (synonymous) changes in single amino acids within this region.

The role of the 3c gene in feline coronavirus replication and pathogenicity is still unknown. However, two studies (3,6) confirmed that a functional 3c protein is required for replication of FECV in the gut epithelium and that FECVs lose their ability to grow in the gut as they gain the ability to grow in macrophages. Positive proof that the 3c protein plays a crucial role in the FIP phenotype will require knowledge of its exact function, of which we currently know very little. A GenBank blast search shows a 30% genetic homology between feline coronavirus 3c and SARS coronavirus 3a (2). Although genetic homology is not pronounced, the 3c protein of feline coronavirus also has an identical hydrophillicity profile to its own membrane (M) protein and to the M and 3a proteins of SARS coronavirus (45). These similarities prompted Oostra and colleagues (45) to state – “(…) it appears that all group 1 [corona] viruses express group-specific proteins predicted to be triple-spanning membrane proteins. Examples are the feline [coronavirus] ORF 3c protein and the HCoV-NL63 ORF 3a protein (…) Despite the small amount of sequence homology among this protein, the similarities in their hydropathy profiles, both to each other and to the corresponding M proteins, as well as to the SARS-CoV 3a protein, are quite remarkable. Nothing is known about these proteins, but it is clear that it will be interesting to learn more about their biological features.” The most recent studies on the 3a protein of SARS coronavirus indicate that it forms a cation-selective channel that is expressed in infected cells and is involved in virus release (46).

Feline coronaviruses, similar to other coronaviruses and to single stranded RNA viruses in general, are highly mutable. A number of different mutant forms of FIPV can coexist in the tissues of cats with FIPV (3,6,7,16). Mutations can be found in many of the structural and accessory genes, although mutations in the large cluster of genes responsible for replication (i.e., the replicase genes) and processing of the virus have been considered to be uncommon (5). A recent study from Cornell University looked at genetic mutations occurring in a specific site of the spike gene that is cleaved by the viral protease (i.e., the S1/S2 cleavage site) (16). Feline coronavirus protease is coded by a gene comprising one of the open reading frames within what is known as the viral replicase. The replicase region codes for several enzymes that are essential for viral replication, including a protease. The S1/S2 cleavage site is where viral protease cleaves the large spike precursor polyprotein to yield functional S proteins. Compared to FECVs, all FIPVs were found to have >1 SNP mutations in and around the S1/S2 cleavage domain. These mutations were unique to each FIPV, and depending on number of mutations and exact amino acids that were changed, they affected the efficiency of the protease to cleave the precursor spike protein. In most cases the efficiency was increased, in some cases decreased, and in other cases not changed. Therefore, it is uncertain how these mutations influence the pathogenesis. However, there was additional evidence suggesting that mutations at this site were important for FECV-to-FIPV conversion. Two cats in this study were shedding FECVs in their feces for two to three years before one of them developed FIP. The S1/S2 cleavage site of FECVs from these two cats and the FIPV from one of them were then compared. Only the fecal FECV from the cat that developed FIPV had a mutation within this region and it was the same mutation that was identified in the FIPV from the diseased tissues. Although this was only one cat, it does suggest that mutations in the S1/S2 cleavage domain of the S gene may be yet another essential element of the FECV-to-FIPV conversion. Because there was not a strong association between mutations in the S1/S2 region observed by the Cornell group and mutations in the fusion domain of the S protein described earlier by the Utrecht group (5, 16), this would constitute a third region involved in the FIPV mutation (two in the S gene and one in the 3c gene). The main conclusion of these various mutation studies is that FECVs undergo a number of mutations at key regions of their genomes as they transition from intestinal epithelial to monocyte/macrophage tropism. Although we still do not understand how these various mutations relate to FIP pathogenesis, they do point to potential targets for antiviral drugs such as protease inhibitors (26) or viral ion channel blockers (46).

A paper published in 2009 (43) temporarily shook the foundations of the internal mutation theory and is still being quoted. Using phyologenetic analyses of a small number (eight) of FIP infected and healthy cats from a New England regional shelter the authors purported to show the existence of two distinct types of feline coronaviruses circulating independently in the population. One population caused FIP and the other did not. The authors based this conclusion on the existence of a five amino acids in the membrane (M) protein of feline coronavirus. They found that certain combinations of these five amino acids were only seen in one “species” or the other. This finding was subsequently refuted by another study that failed to confirm these findings and again concluded that FIPVs originate from FECVs in the same environments (44). This study tested far more cats and compensated for differences in the origins of their isolates by comparing FIPVs with FECVs from cats that shared the same environments. Additional studies on a large population of cats, and also taking into consideration genetic differences related to geographic origins, also failed to find evidence for the “two virus” theory or any relationship between certain M gene differences and FIP vs enteric biotypes (3).

FECV infection studies

Two studies from different groups were concerned with the behavior of FECV infection and immunity in laboratory cats (7,8). These experiments confirmed what had been previously observed from cats naturally infected with FECVs. Primary FECV infection is largely asymptomatic and is centered in the lower small bowel and colon (8,9). Large amounts of virus are shed in the feces for many weeks, and even months after initial infection, but with time most cats stop shedding. There also appears to be a low level of FECV in blood monocytes during initial infection (9). However, immunity is not always solid and as antibody levels in the blood drop many cats become susceptible to reinfection (7). These secondary infections closely resemble the primary infection.

FECVs, and therefore FIPVs, exist in two forms or serotypes. Serotype 1 FECVs/FIPVs are unique to cats, while serotype 2 viruses are formed from recombination between spike genes of type 1 FECVs and closely related canine coronaviruses. Type 1 FECVs/FIPVs predominate in Europe and the Americas, while up to 25% or more of isolates from Asian countries are type 2 (10-13). Type 2 FIPVs appear to be more virulent than type 1 FIPVs (11,14).

Even though numerous strains of FECV are found among cats around the world, at least one population has remained surprisingly free. Cats in the Falkland Islands have no signs of infection and attempts are being made to quarantine these cats from an inadvertent introduction of the virus (15).

FIPV transmission (vertical vs horizontal)

The issue of whether FIPV is transmitted cat-to-cat (horizontal transmission) or by internal mutation from FECV (vertical transmission) has also been a topic of interest. There is no solid evidence that cats with FIP can transmit FIPV directly to other cats, although the possibility has been evoked as a possible explanation for rare mini-epizootics of FIP (1). FIPV can cause disease when fed to laboratory cats, and some experimentally infected cats will shed low levels of FIPV-like virus (2,3). However, this virus does not appear to be infectious when fed to other cats (3). Some cats with naturally acquired FIP may also shed either FECV or a coronavirus that appears genetically similar to the virus in the body of a cat with FIP (6). An explosive outbreak with a serotype 2 FIPV in Taiwan does appear to result from some sort of horizontal transmission (14). Nonetheless, cats with FIP are not considered to be highly infectious to other cats and the most likely route of transmission still appears to be via FECV and internal mutation.

Immune and immunopathologic responses in FIPV infection

A number of recent studies have dealt with the immune response to FIPV. It has been known for some time that FIPV infection causes a depletion of lymphocytes, mainly T cells. More specifically, it has been shown that natural killer (NK) and regulatory T (Treg) cells are greatly depleted in the blood of cats with FIP (17). This would result in a reduced capacity of the innate immune system (NK cell mediated) to destroy infected macrophages and a depressed ability to suppress the resultant inflammatory responses (Treg mediated). Both of these are typical features of FIP. At the same time that innate and cell mediated immune responses (components of T cells) are being depleted, B cells involved in antibody mediated immunity are being stimulated (18). A number of cytokine mediators associated with B cells were increased in cats with FIP, suggesting that virus infected macrophages overproduce B-cell differentiation and survival factors and thus promote the humoral side of the immune response. It is well known that humoral immunity may actually enhance virus infection and replication in macrophages and contribute to the antibody/antigen/complex mediated vasculitis that is characteristic of the disease (reviewed 1). This same research group also found that vascular endothelial growth factor (VEGF), produced by feline infectious peritonitis (FIP) virus-infected monocytes and macrophages, induces vascular permeability and may be an important contributor to abdominal and thoracic effusions in cats with wet FIP (19). Monocytes and neutrophils, which are dominant cells in both the fluids and lesions, were found to have increased expression of adhesion molecules while in the bloodstream (20). Adhesion molecules are critical for these cells to stick to the endothelium in areas of inflammation and then migrate through the gaps between endothelial cells and into the surrounding tissues.

Are genetic factors involved in FIP susceptibility?

Genetic factors are presumed to influence the incidence of feline infectious peritonitis (FIP), especially among pedigreed cats. However, proof for the existence of such factors has been limited to Persians and somewhat anecdotal. A recent genome wide association study (GWAS) has strongly implicated a number of regions of the genome of Birman cats dying of FIP with susceptibility (41). DNA from 38 Birman cats that died of FIP and 161 healthy cats from breeders in Denmark and USA were selected for GWAS. Birman cats were chosen for the study because they are highly inbred and suffer a high incidence of FIP. This study found Danish and American Birman to be closely related and the populations were therefore combined and analyzed in two manners: (1) all cases (FIP) vs. all controls (healthy) regardless of age, and (2) cases 1½ years of age and younger (most susceptible) vs. controls 2 years of age and older (most resistant). GWAS of the second cohort was most productive in identifying significant genome-wide associations between case and control cats. Four peaks of association with FIP susceptibility were identified, with two being identified on both analyses. Five candidate genes ELMO1, RRAGA, TNFSF10, ERAP1 and ERAP2, all relevant to what is known about FIP virus pathogenesis, were identified but no single association was fully concordant with the disease phenotype. However, none of these genes were sequenced and mutations associated with susceptibility confirmed and no test for susceptibility is therefore available. However, this study confirms the wisdom of not using any male or female for breeding that have produced kittens that died from FIP, siblings of cats that have died of FIP, or any offspring or sibling of a cat that has died of FIP.

Diagnostic tests for FIP

Classic tests for FIP include the complete blood count (CBC), albumin and globulin levels (and A:G ratio), and basic blood chemistries (reviewed 1). The characteristic findings of these tests include a chronic unresponsive anemia (anemia of chronic disease), a high white cell count with an absolute increase in neutrophils and an absolute decrease in lymphocytes, high globulin, low albumin, low A:G ratio, hyperbilirubinuria. Although many cats with FIP exhibit all of these changes, it is best to go by the 70% rule. Any one of these abnormalities will occur in at least 70% of cats with FIP. If these changes are coupled with other risk factors, in particular age under 18 months, origin from a cattery, shelter or rescue type facility, and disease signs such as abdominal distension, dyspnea from pleural effusion, neurologic signs or uveitis, a diagnosis can be made with high certainty.

The presence of a characteristic type fluid in the abdomen or less frequently the chest is one of the most diagnostic features of the wet form of FIP. Wet FIP predominates in most breed and random bred cats, except for Birman and Burmese, which suffer predominantly from the dry form. The fluid is usually yellow tinged (rarely clear or green-tinged), mildly to notably cloudy, viscous (egg-white consistency), high in protein and containing many cells including macrophages, neutrophils and lymphocytes. The fluid will often contained visual bits of fibrin and will frequently form a partial clot when let sit in a tube without anti-coagulants. As explained above, it is usually possible to detect the FIPV in the fluid by PCR or by immunohistochemistry (see below). The exudate of FIP is unlike that seen in rare cases of bacterial peritonitis. This fluid is clearly purulent in appearance, may have a fetid odor, and is not viscous. Transudates and modified transudates associated with cancers do not have the same physical and cellular characteristics of FIP fluids.

The ratio of albumin to globulin ratio (A:G) has been touted as a useful predictor of FIP (reviewed 1). Albumin levels often drop during chronic diseases and globulins rise when there is a chronic infection. Therefore, it is not surprising that A:G decreases in most cats with FIP. However, the predictive value of the A:G ratio is very much like other laboratory abnormalities that are frequently associated with FIP. The more other features of FIP are present, such as young age, shelter or cattery origin, ascites, uveitis, CNS disease signs, etc., the more likely a low A:G will confirm FIP as the cause. In a study of FIP cases seen in a mid-western referral practice, it was concluded that when the prevalence of FIP is low, a low A:G is more useful to rule out FIP but is not helpful in making a positive diagnosis of FIP (42). The opposite is true when the prevalence of FIP is high and/or a number of other FIP risk factors are present.

Controversy still abounds over the use and interpretation of feline coronavirus antibody titers in the serum (reviewed 1). The main problem with antibody tests is that both FECVs and FIPVs, being virtually identical to each other, evoke the same antibody responses. The fact that FECV infection is ubiquitous among cats, and especially among catteries and shelter-type environments that yield most FIP cases, means that many healthy cats will also be antibody positive. Feline coronavirus antibody titers, if accurately done, are nonetheless of some value. Although many healthy FECV exposed cats have titers from 1:100 to 1:400 (7), as do many cats with FIP, the likelihood of a titer being associated with FIP increases with the magnitude of the titer. Few healthy cats have titers of 1:1600 and titers of 1:3200 and above are highly indicative of FIP. Feline coronavirus titers also have some value in predicting whether a cat may be shedding FECV in their stool. Hardly any cat with a titer of 1:25 and lower are fecal shedders, while almost all cats with titers of 1:400 and above are shedding (7). There have been attempts to make antibody tests more specific for FIPV, just as there have been attempts to make PCR-based tests more FIPV specific. A classical test, which is still available, is the 7b antibody test. This test was developed on a faulty premise that FECVs lack the 7b gene and hence do not produce 7b protein, while FIPVs have the 7b gene, produce the protein, and therefore also evoke an antibody response to 7b. It is now well known that all legitimate FIPVs and FECVs possess an intact 7b gene and therefore evoke 7b antibodies. This fact as it relates to the 7b antibody test was reconfirmed by a recent study. Ninty five serum samples submitted for various diagnostic assays and 20 samples from specific-pathogen-free cats (free of coronavirus infection and antibodies) were tested for antibodies to purified 7b protein (32). As expected, expression of the 7b protein, as indicated by detection of antibodies against the protein, was found in most feline coronavirus (i.e., FECV or FIPV) infected cats and it was concluded that seropositivity for this protein was not specific for FIPV or a diagnosis of FIP. In essence, the 7b antibody test is no different than regular feline coronavirus antibody tests.

The Rivalta test is widely touted, especially in Europe, for diagnosing FIP-associated exudates. The test involves placing a few drops of ascitic or thoracic fluid into a tube containing a weak acetic acid solution. The appearance of a white flocculent material is seen in a positive test. A positive Rivalta test was once believed to be highly specific for FIP fluid. However, recent studies looking at its application to a much wider range of cats with effusions has noted that its sensitivity and specificity were lower than previously reported, except in young cats (£2 yrs) with effusions and with cases of lymphoma or bacterial infections excluded(27). This is logical because the specificity and sensitivity of any indirect test for FIP will increase greatly when applied to cats at the highest risk for the disease or having other FIP-related test abnormalities. For example, an anemia of chronic disease in a young cat would be far more indicative of FIP than the same signs in an aged cat. This same study demonstrated that protein levels alone could not explain the Rivalta reaction.

Several studies have involved various diagnostic tests for FIP, all claiming to assure a more ready and accurate diagnosis. The gold standard for all diagnostic tests is the identification of FIPV within diseased tissues or fluids. A real time PCR test was able to detect viral RNA in 62% of ascites samples from purebred cats and from 35% of crossbreds presumed to have FIP (10). The detection rate was 82% in Norwegian Forest cats and 78% in Scottish folds, which was higher than any other breeds tested. This study was limited in that a diagnosis of FIP was not confirmed after testing so it is difficult to interpret the results. A real time PCR test, if properly configured and run, should be able to detect most infections. Therefore, the fact that only 62% of overall ascites samples were positive suggests either that cats with diseases other than FIP were being sampled or more likely that the actual testing was insensitive or inhibitors of the PCR reaction were present. RT PCR is quite accurate in detecting and semi-quantitating fecal coronavirus shedding in both experimental and naturally infected cats (2,3,7-16).

Immunostaining of diseased tissues (immunohistochemistry) or fluids by either immunofluorescence or immunoperoxidase methods has probably been a more reliable method overall than even PCR, but the accuracy is limited by the quality of the material that is sampled and of the test procedure. Its usefulness also depends on the ability to obtain the necessary samples either premortem or antemortem. Immunoperoxidase was used to diagnose FIPV in macrophages in the skin of two cats with atypical skin lesions (multiple popular lesions) (21,33) and in various tissues of a diseased Mountain lion (22). The technique has also been used successfully to detect FIPV infected macrophages within the cerebrospinal fluid of a cat with neurologic disease (23). Immunohistochemistry on cells from abdominal or thoracic effusions should be used much more often in cats suspected of having FIP. Immunohistochemistry, when it can be employed, is considered to be one of the most definitive tests among many that are currently used (31).

FIP therapeutics

Several approaches have been used to treat cats with FIP. Most potential therapies are targeted to either the inflammation induced by the infection or to the virus itself. A third approach is to non-specifically stimulate the immune system on the hopes that it will be able to overcome the infection.

Cyclosporin A, a potent immunosuppressive drug, has been shown to inhibit feline coronavirus replication in cell cultures (24). The drug chloroquine, which is used to treat malaria, has also been shown to inhibit FIPV replicaton in cell cultures and may have had some effect on improving clinical scores in cats with FIP (25). However, it appeared to be hepatotoxic. The problem with non-specific viral inhibitors of this type is that they are inhibitory of viral replication in the test tube but not in the cat.

Tumor necrosis factor (TNF) inhibitors have been used for some time to alleviate some of the signs of FIP. One of the most popular of these drugs is pentoxyfilline (29). Pentoxyfylline was widely used in FIP cases because of its use in controlling vasculitis in humans, and vasculitis is an important component of the pathophysiology of FIP. A recent study with 23 cats with proven FIP failed to detect an effect of pentoxyfylline on the survival time, the quality of life, or any FIP associated clinical or laboratory parameters of FIP (29).

One of three cats treated with a biologic called polyprenyl immunostimulant (PI) were reportedly cured of FIP after long term treatment (28). However, all three cats had disease localized to a single mesenteric lymph node and two were asymptomatic and the third appeared only a little rough. The treatment had no effect on cats with more severe disease, such as wet FIP. Subsequent trials with the drug, which excluded cats with wet FIP and other severely ill cats (presumably with dry FIP) showed only a 5% survival at one year. Although the study was not double blinded and placebo controlled, claims have been made that PI treatment, while not curing cats of FIP, may prolong their lives. PI is an expensive and ineffective treatment for FIP, even if merely for prolonging life.

The most promising experiments have involved drugs that specifically target certain protein activities of feline coronaviruses. Feline coronaviruses have several genes that are similar in activity to those of HIV, including a reverse transcriptase and a protease. Reverse transcripase (RT) is needed by the virus to convert its RNA form to copy DNA (cDNA), then to messenger RNA (mRNA) and finally to specific proteins. RT inhibitors play an important role in HIV treatment known as highly active anti-retroviral therapy. Unfortunately, there are no RT inhibitors for coronaviruses at the present time. However, promising inhibitors of the viral protease are now being developed (26). Protease is an enzyme encoded by the viral protease gene and responsible for cutting viral proteins into their final form, which is necessary for assembly of new viruses. Many of the structural proteins of coronavirus are first transcribed from mRNA as what are called polyproteins. Polyproteins must be cut by protease into their constituent parts.

FIP in other animal species

FIP has been well documented to occur in virtually every species of wild felids. Although described previously and in passing in the Mountain lion (Puma), a detailed report of FIP in a California Mountain lion has been recenty published (22).

Cheetahs and FIP have an interesting connection. Cheetahs are highly inbred as a result of severe bottlenecks that have occurred in the population over thousands of years. It has also been shown that cheetahs will often accept skin grafts from each other, again suggesting that they are closely related. The highly inbred nature of cheetahs could theoretically leave them vulnerable to an infectious agent to which they cannot respond and wipe them out as a species. An outbreak of FIP among captive Cheetahs in Oregon several decades ago was seen as a fulfillment of this prophecy. However, after the initial outbreak, further FIP loses subsided. This would be expected if feline coronavirus infection (i.e., FECV infection) of Cheetahs followed the same course as occurs in cats. In this scenario the infection would switch from an epizootic to an enzootic state and the population of cheetahs would adjust to the new virus as if they were domestic cats. Recent studies have confirmed this scenario. Cheetahs in the wild have an extremely low rate of exposure to many common cat infections including feline coronavirus and rarely suffer infectious diseases (40). However, almost one-half of healthy captive cheetahs shed feline coronavirus in their feces persistently, transiently, or intermittently (41) and FIP losses after the initial outbreak remain very low. Although highly inbred, cheetahs in captivity appear to be no more susceptible to FIP than some breeds of cats.

An epizootic of catarrhal enteritis caused by a novel coronavirus was first described in pet Ferrets in the US in 1993 (reviewed 34). Subsequently, a disease identical in appearance to dry FIP of cats was also described in ferrets in the US (34). This same disease has subsequently been reported in pet ferrets in Japan and Europe (35,37,38). A genetic analysis of several additional isolates from ferrets with enteritis or FIP-like disease suggested that the causative agents of the two diseases were closely related but genetically unique (36). Interestingly, functional mutations resembling those of FIPV were seen in two of three isolates from ferrets with FIP-like disease but in none of the isolates from ferrets with enteritis. These viruses in ferrets would appear at firsthand to resemble FECVs and FIPV in cats and whether viruses causing FIP-like disease and enteritis in ferrets are different viruses or one is a mutation of the other remains to be determined from studies of more isolates. What is certain is that ferret infectious peritonitis is a relatively new and increasing problems in pet ferrets and is hauntingly similar to the appearance of FIP for the first time in cats in the late 1950s (reviewed 1). The fact ferret coronavirus exists in many genetic forms indicates that it coronaviruses have been in the species for some time. Has a new ferret coronavirus evolved that is more likely to mutate to cause infectious peritonitis? As ferrets have become more popular as pets, do they face the same risk factors that faced cats as they became more popular as pets?

SOCK FIP CORE TEAM MEMBERS

WHY IS SUPPORT NEEDED NOW?

Although there have been over 500 clinical and research publications on FIP since 1963, solutions to the disease have eluded researchers. However, that situation is now changing:the sequencing of the entire feline genome is almost complete and will provide researchers with a set of tools of unprecedented power to answer some of the important remaining questions about FIP.

HISTORY OF SOCK

In 1974, Save Our Cats and Kittens (SOCK) was organized as a nonprofit group.It was founded by cat lovers from the San Francisco Bay Area to aid in much-needed feline leukemia virus (FeLV) research at the UC Davis School of Veterinary Medicine.FeLV infection was the cause of death of up to one-third of all sick cats during the 1960s and 1970s.